Mucus obstruction is a key pathogenic step in many major human ainways diseases, including COPD and CF. As part of the drug discovery process, it is extremely valuable to have a small animal model that will accelerate the transition from in vitro studies of target identification/validation to eariy proof-of-concept studies in humans. The (3ENaC mouse has been engineered to exhibit many of the features of muco-obstructive human airways diseases, expressing a disease-initiating transgene (PENaC) that drives ainA/ay surface dehydration. The ainways dehydration of the PENaC mouse triggers a sequence of intraluminal airways mucus plaque/plug formation, airways inflammation with macrophage activation, ain/vays remodeling, bacterial infection, and emphysema. In Project II, we propose to contribute to the tPPG goals and needs for drug development as follows: 1) test in vivo the novel biophysical/biochemical formulation of a two-gel mucus clearance system, focusing on relationships between the rate of delivery of hypertonic saline (HS) and mucus clearance responses; 2) develop novel measures of mucus properties that will serve as validated biomarkers to predict the magnitude of the ain/vay mucus burden, i.e., our therapeutic target, utilizing PENaC mouse lines of differing ainways Na+ transport/ainway surface dehydration; 3) test the hypothesis that secreted mucins (MUC5AC, MUCSB) are therapeutic targets with favorable risk:benefit ratios by genetic studies of MUC5AC and MUCSB -/- crosses with PENaC mice and pharmacologic (novel ketolides/macrolides) approaches; and 4) test the hypothesis that bacterial infection of mucus-obstructed ainways can be prevented or reversed by hydration/mucolytic therapies. Thus, Project II should provide important in vivo data as to the validity of our novel two-gel model of mucus transport, identify biomarkers for pharmacodynamic studies of mucus modification to speed drug development in both animals and the clinic, and assist Core A to identify relevant targets and novel therapies of muco-obstructive lung disease.